i) Field of the Invention
The present invention relates to a quantitative determination of pyruvic acid and a quantitative analysis for a component of a living body making use of such a determination.
ii) Description of the Background Art
In the field of clinical chemistry, there are generally used processes for the quantitative analysis for a component of a living body in which the component of the living body is oxidized in the presence of an oxidase to form hydrogen peroxide, and the resulting hydrogen peroxide is converted into a quinone type pigment with a peroxidase and a color former, thereby conducting colorimetry, and in which an oxidation-reduction reaction between oxidized nicotinamide adenine dinucleotide (hereinafter referred to as "NAD") and reduced nicotinamide adenine dinucleotide (hereinafter referred to as "NADH") by a dehydrogenase is used to determine increase or decrease in the absorbance of NADH at a wavelength of 340 nm.
When the amount of pyruvic acid existing in a living body or formed in the course of a biological reaction is determined, it is known to oxidize pyruvic acid with a pyruvate oxidase to form hydrogen peroxide and then convert hydrogen peroxide thus formed into a quinone type pigment with a peroxidase and a color former, thereby conducting colorimetry. It is also known to react a lactate dehydrogenase and NADH with pyruvic acid existing in a living body or formed in the course of a biological reaction and determine decrease in the absorbance of NADH at a wavelength of 340 nm.
The process in which hydrogen peroxide formed by the pyruvate oxidase is determined however involves problems such that a negative error is produced by reducing substances among the components of the living body, for example, ascorbic acid, bilirubin and the like, and when the concentration of pyruvic acid is high, dissolved oxygen in a reaction reagent becomes insufficient, resulting in a failure in determination.
On the other hand, the process in which the lactate dehydrogenase is used to determine decrease in the absorbance of NADH at a wavelength of 340 nm can widen the measuring range in dependence on the concentration of NADH, but involves a problem that when the concentration of NADH is increased, an initial absorbance is also increased, so that the measurement of the absorbance becomes impossible from the viewpoint of the performance of a spectrophotometer.